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Volume 7 issue 2 February 2006
The inner tegument promotes herpes simplex virus capsid motility along microtubules in vitro
André Wolfstein, Claus-Henning Nagel, Kerstin Radtke, Katinka Döhner, Victoria J. Allan and Beate Sodeik
SUPPLEMENTARY MATERIAL:
Movie S1. HSV1-GFPVP26 capsid motility along MT was reconstituted in vitro .
This movie (left panel) corresponds to the tracks 19 to 22 and 57 in Fig. 1 (c.f. left insert). HSV1-GFPVP26 capsids (green dots) were purified from extracellular virions after extraction with 1% Triton X-100 and 1 M KCl. They were resuspended in cytosol with a protein concentration of 2.5 mg/ml, and perfused into a microscopic observation chamber that had been pre-coated with Cy3-labelled MT (red). Several microscopic fields corresponding to an area of 48.3 µm by 64.9 µm (c.f. Fig. 1) were recorded for 320 seconds at a temporal resolution of 1.6 seconds per frame using the GFP-filter and a 100x objective. The GFP-sequence was superimposed over one image taken with the Cy3 filter set. Scale bar (yellow): 5 µm. Translocations of capsids in this movie are displayed as track profiles (right panel). Arrowheads indicate the direction of translocation.
Movie S1 (.mov); Movie S1 Still (.tif); Movie S1Track (.tif)
Movie S2. Variation in velocity of HSV1-GFPVP26 capsid motility along MT in vitro .
HSV1-GFPVP26 showed different types of behaviour in the in vitro assays. The asterisk indicates a capsid that stably bound to MT, and the movie shows an example of a fast capsid (lower arrowhead) moving along Cy3-labelled MT with an average velocity of 0.13±0.02 µm/seconds and a peak velocity of 1 µm/seconds, and another example for a slow transport along Cy3-labelled MT (right arrowhead) with an average of 0.10±0.00 µm/seconds and a peak velocity of 0.26 µm/seconds.
HSV1-GFPVP26 capsids (green dots) were purified from extracellular virions after extraction with 1% Triton X-100 and 1 M KCl. They were resuspended in cytosol with a protein concentration of 10 mg/ml, and perfused into a microscopic observation chamber that had been pre-coated with Cy3-labelled MT (red). A microscopic field was recorded for 155 seconds at a temporal resolution of 1.6 seconds per frame using the GFP-filter and a 100x objective and a digital camera. The GFP sequence was subsequently superimposed over one Cy3 image taken after the movie. Scale bar (yellow): 5 µm. Translocations of capsids in this movie are displayed as track profiles (right panel). Arrowheads indicate the direction of translocation.
Movie S2 (.mov); Movie S2 Still (.tif); Movie S2 Track (.tif)
Movie S3. Long-distance HSV1-GFPVP26 capsid transport along MT in vitro .
HSV1-GFPVP26 showed different types of behaviour in the in vitro assays. This movie (left panel) shows an example of a fast transport that starts on the left (left arrowhead) with an average velocity of 0.31±0.21 µm/seconds over a long distance of 30.5 µm. Another rather short transport starts on the right (right arrowhead).
HSV1-GFPVP26 capsids (green dots) were purified from extracellular virions after extraction with 1% Triton X-100 and 1 M KCl, resuspended in cytosol with a protein concentration of 5 mg/ml, and perfused into a microscopic observation chamber that had been pre-coated with Cy3-labelled MT (red). A microscopic field was recorded for 128 seconds at a temporal resolution of 1.6 seconds per frame using the GFP-filter and a 100x objective. The GFP sequence was subsequently superimposed over one Cy3 image taken after the movie. Scale bar (yellow): 5 µm. Translocations of capsids in this movie are displayed as track profiles (right panel). Arrowheads indicate the direction of translocation.
Movie S3 (.mov); Movie S3 Still (.tif); Movie S3 Track (.tif)
Movie S4. HSV1-GFPVP26 capsid transport in vitro along Cy3- and unlabelled MT.
This movie (left panel) shows examples of HSV1-GFPVP26 capsid motility along Cy3-MT (yellow arrowhead), and active transport across a MT with a labelling efficiency too low to be detected (yellow arrow). The capsid switched from the unlabelled to the Cy3-MT, and later paused before switching direction on the Cy3-MT.
HSV1-GFPVP26 capsids (green dots) were purified from extracellular virions after extraction with 1% Triton X-100 and 1 M KCl. They were resuspended in cytosol with a protein concentration of 2.5 mg/ml, and perfused into a microscopic observation chamber that had been pre-coated with Cy3-labelled MT (red). Capsid motility was recorded for 304 seconds at a temporal resolution of 1.6 seconds per frame using the GFP-filter and a 100x objective. The GFP sequence was subsequently superimposed over one Cy3 image taken after the movie. Scale bar (yellow): 2 µm. The translocation of the capsid in this movie is displayed as track profile (right panel). The arrowhead indicates the direction of translocation.
Movie S4 (.mov); Movie S4 Still (.tif); Movie S4 Track (.tif)
Movie S5. HSV1-GFPVP26 tracks along MT in vitro were often interrupted by pauses.
HSV1-GFPVP26 showed different types of behaviour in the in vitro assays. This movie (left panel) shows an example of a 6 seconds pause which a capsid (yellow arrowhead) took between two consecutive runs. This capsid moved with an average velocity of 0.15±0.02 µm/seconds and a maximal velocity of 1 µm/seconds along a track of 17.7 µm length.
HSV1-GFPVP26 capsids (green dots) were purified from extracellular virions after extraction with 1% Triton X-100 and 1 M KCl. They were resuspended in cytosol with a protein concentration of 5 mg/ml, and perfused into a microscopic observation chamber that had been pre-coated with Cy3-labelled MT (red). A microscopic field was recorded for 166 seconds at a temporal resolution of 1.6 seconds per frame using the GFP-filter and a 100x objective. The GFP sequence was subsequently superimposed over one Cy3 image taken after the movie. Scale bar (yellow): 5 µm. The translocation of the capsid in this movie is displayed as track profile (right panel). The arrowhead indicates the direction of translocation.
Movie S5 (.mov); Movie S5 Still (.tif); Movie S5 Track (.tif)
Table S1. Statistical analysis of HSV1-GFPVP26 capsid motility along MT in vitro .
Top to bottom: influence of cytosol concentration (c.f. Fig. 2b), dynamitin (c.f. Fig. 2c, d), and tegument protein composition (c.f. Fig. 5) on capsid motility. For each experiment, the track length (complete translocated distance d from beginning to end of recording; for definition c.f. Fig. 1a, left insert), run length (translocated distance between two pauses), the number of runs per track, pause length, translocated distance between two consecutive frames (d/frame), and velocity and their respective SEM (standard error of the mean) are listed. The number of events (n) for each parameter is listed in brackets behind. The P values were calculated in two-sided Student t-tests assuming unequal variances. Significant P-values were indicated with one red asterisk and highly significant P-values with two. Experiments were conducted on a Leica DM IRB/E microscope with a 100x objective. Temporal resolution of the experiments is influenced by the microscopic setup used in a given experiment, the mean values of parameters varied accordingly.
All particles are the mean of all particles in the focus plane counted in the first and in the last frame in all recorded movies. The relative number of motile particles is based on all particles . MT density was calculated as described in supplementary figure S1.
Table S1 (.doc)
Table S2. Antibodies used in this study.
Table S2 (.doc)
Figure S1. Stereological Analysis of MT length.
A randomly oriented lattice with a line spacing D of 55 Pixel was superimposed on the MT-image (A) and rotated around three random angles. The number of intersections of lattice and MT is proportional to MT density. The grey values of all pixels of the MT image were converted into an MS Excel datasheet (B) with the TILLVision software and analyzed for intersections with the lattice via a visual basic program based on MS Excel. The high grey values of the MT are given as red numbers (C, datasheet B at larger magnification). Some intersections are marked with arrows. Total MT length L was calculated according from the number of intersections N as follows: L [µm] = N x D x pixel size x magnification.
Figure S1 (.tif)
Figure S2. The cytosol preparation is free of membranes.
To test whether the cytosol prepared from Xenopus egg extract by centrifugation contained any residual membranes, Xenopus extract was incubated with 1 µM Dil, a fluorescent membrane dye, prior to the centrifugation step. The resulting pellet (A; bar: 20 µm) was resuspended in buffer and analyzed by fluorescence microscopy together with the cytosolic supernatant (B). Buffer incubated with Dil was used as a control (C). Only the pellet contained Dil-labeled membranes.
Figure S2 (.tif)
Figure S3. Viral and nuclear capsids differ in their tegument protein composition.
Extracellular HSV1, strain F virions were not treated (no lysis), or lysed with 1% (v/v) Triton X-100 in the presence of different KCl concentrations (0.1, 0.5 or 1 M) to generate viral capsids of different protein composition. Virus lysates were fractionated by centrifugation through a 20% (w/v) sucrose cushion. Nuclear B and C capsids were isolated by extracting infected nuclei. Intact virions (no lysis), the pellets derived from fractionated virions (0.1, 0.5, 1), nuclear capsids (B, C), and the supernatants (0.1, 0.5, 1) were subjected to SDS-PAGE and immunoblotting (see Supplementary table S2, online for information on the antibodies). For comparison, nuclear capsids were used at higher concentration than the other samples as shown by the amount of the capsid proteins VP5, VP19c and VP26.
Figure S3 (.tif)
Supplemental references:
References (.doc)
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